Terminal differentiation is often coupled with permanent exit from the cell cycle and represents the most common cellular state in adult animals. Yet it remains unclear how proliferation is blocked in differentiated tissues. The goal of this project is to determine how terminal differentiation signals impinge on the cell cycle machinery to induce a stable quiescent state and investigate how this state is disrupted in cancer. In the research proposed here, I use a combination of genetic and biochemical approaches in Drosophila and mammalian cells to delineate the conserved genetic pathways that control cell cycle exit. During the K99 phase of this grant, I investigated the redundant mechanisms that limit cycling in differentiated Drosophila tissues, obtained experience working with 2 different mammalian cell lines that can be induced to differentiate in vitro for future studies of cell cycle exit, and worked on a screen to identify genes that when overexpressed or inhibited by RNAi, de-regulate the cell cycle to cause ectopic proliferation in contexts of terminal differentiation. Such genes would normally be activated or inhibited upon terminal differentiation, but likely become de-regulated in cancers. During the ROO phase of the award, I will determine the precise mechanisms by which these genes regulate the cell cycle (Aim 1) and examine the roles of mammalian orthologs of these genes in normal and cancerous mammalian cells (Aim2). I will also investigate the signaling pathways by which these genes are regulated, using genetic epistasis tools in both Drosophila and mammalian cells (Aim 3). Such work will begin to delineate the conserved pathways connecting terminal differentiation signals with cell cycle controls, and determine how they can become de-regulated leading to cancer.